Currently a vaccine is available which can be used to immunize susceptible persons against infection by hepatitis B virus. This vaccine is derived by purification of the spherical 22 nm hepatitis B surface antigen (HBsAg) from the plasma of humans who have been infected by hepatitis B virus and who have become chronic producers of surface antigen.
The human plasma-derived HBsAg is recoverable as a 22 nm particle containing both a glycosylated polypeptide (reported as having a molecular weight of 27,000-28,000 daltons) and a nonglycosylated polypeptide (reported as having a molecular weight of 23,000-26,000 daltons). This polypeptide is coded for by the HBsAg gene. Human plasma-derived HBsAg contains cholesterol and is free of phosphatidylinositol. It is stable at pH 2 and in the presence of pepsin at pH 2. It is stable also when treated with HCHO, and then with dithiothreitol and sodium dodecyl sulfate (SDS), followed by boiling for 15 minutes. After such treatment plasma-derived antigen does not migrate into an SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. Covalent disulfide bonds exist between essentially all monomeric protein molecules of human plasma derived HBsAg. The immunogenicity of human HBsAg and thus the ability of the human HBsAg to be used as a vaccine is known to be critically dependent on the disulfide bonded form of the antigen (Mishiro et al., J. Immunol., 124: 1589, 1980). If few disulfide bonds of HBsAg are present then the immunogenicity of the HBsAg is drastically reduced.
In order to insure the safety of the vaccine derived from human plasma, multiple steps are required in the purification process in order to inactivate potential contaminating infectious agents. Although this human derived vaccine has been useful, investigators have sought an alternate source for immunogenic 22 nm particles. The major approach to an alternate source has been synthesis of HBsAg in E. coli, Saccharomyces cerevisiae or mammalian cells using molecular cloning techniques. The gene for HBsAg has been cloned and sequenced. However, limited success has been achieved in expression of the cloned gene. The 22 nm HBsAg apparently is difficult to make in a prokaryotic organism. The synthesis of HBsAg in Saccharomyces cerevisiae has been reported by Valenzuela et al., Nature, 298, 347-350 (1982), by Hitzeman et al., Nucleic Acid Research, 11, 2745-2763 (1983) and by Miyanohara et al., PNAS 80, 1-5 (1983).
Hitzeman et al., supra, disclosed that 2-5% of the HBsAg polypeptide obtained upon yeast cell disruption is found in aggregated form similar in size, density and shape to the 22 nm particle isolated from the plasma of human hepatitis carriers. However, the nature of the interaction between the monomeric subunits in this 22 nm particle has not been investigated.